Scrambling of Sequence Information in Collision-Induced Dissociation of Peptides

Department of Chemistry, University of Toronto, Toronto, Ontario, Canada
Journal of the American Chemical Society (Impact Factor: 12.11). 09/2006; 128(32):10364-5. DOI: 10.1021/ja062440h
Source: PubMed


Collision-induced dissociation (CID) of protonated YAGFL-NH2 leads to nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicate that primary fragmentation of the intact peptide leads to the linear YAGFLoxa b5 ion with a C-terminal oxazolone ring that is attacked by the N-terminal amino group to induce formation of a cyclic peptide b5 isomer. The latter can undergo various proton transfer reactions and opens up to form something other than the YAGFLoxa linear b5 isomer, leading to scrambling of sequence information in the CID of protonated YAGFL-NH2.

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    • "[14] Recently, numerous experimental and theoretical studies have revealed that middle-sized b n (n = 5, 6, 7 …) ions undergo intramolecular head-to-tail cyclization to form a protonated cyclic peptide intermediate. [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] Upon proton transfer (PT), this cyclic structure subsequently reopens at different amide bonds to form a variety of C-terminal oxazolone b isomers with a permuted sequence, which may result in erroneous assignment of fragment ions for database searches. The lysine acetylation is one of the most prominent posttranslational modification found in proteins and plays vital roles in regulation of protein activity and gene expression. "
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    ABSTRACT: Characterization of ε-N-acetylated lysine containing peptides, one of the most prominent post-translational modifications of proteins, is an important goal for tandem mass spectrometry experiments. A systematic study for the fragmentation reactions of b ions derived from ε-N-acetyllysine containing model octapeptides (KAc YAGFLVG and YAKAc GFLVG) has been examined in detail. Collision-induced dissociation (CID) mass spectra of bn (n = 4-7) fragments of ε-N-acetylated lysine containing peptides are compared with those of N-terminal acetylated and doubly acetylated (both ε-N and N-terminal) peptides, as well as acetyl-free peptides. Both direct and nondirect fragments are observed for acetyl-free and singly acetylated (ε-N or N-terminal) peptides. In the case of ε-N-acetylated lysine containing peptides, however, specific fragment ions (m/z 309, 456, 569 and 668) are observed in CID mass spectra of bn (n = 4-7) ions. The CID mass spectra of these four ions are shown to be identical to those of selected protonated C-terminal amidated peptides. On this basis, a new type of rearrangement chemistry is proposed to account for the formation of these fragment ions, which are specific for ε-N-acetylated lysine containing peptides. Consistent with the observation of nondirect fragments, it is proposed that the b ions undergo head-to-tail macrocyclization followed by ring opening. The proposed reaction pathway assumes that bn (n = 4-7) of ε-N-acetylated lysine containing peptides has a tendency to place the KAc residue at the C-terminal position after macrocyclization/reopening mechanism. Then, following the loss of CO, it is proposed that the marker ions are the result of the loss of an acetyllysine imine as a neutral fragment. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.
    Journal of Mass Spectrometry 12/2014; 49(12):1290-7. DOI:10.1002/jms.3462 · 2.38 Impact Factor
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    • ". The possible reopening of the cyclic peptide at several positions can lead to scrambling of the initial amino acid sequence and thus lead to a complex variety of oxazolones [21] [25] [31] [34]. Further fragmentation of these scrambled b-ions leads to fragment ions (a-ions or lower b-ions) that are hard to assign to the initial peptide [30] [31]. Quantifying the proportion of b-ion cyclization is still object of recent research [36]. "
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    ABSTRACT: Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16 % of b-ion loss.
    Biochimica et Biophysica Acta 08/2013; 1834(12):2843-8. DOI:10.1016/j.bbapap.2013.08.007 · 4.66 Impact Factor
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    • "It was also postulated that protonated ␣-amino␧-caprolactam was formed via involvement of a lysine side chain ␧-amine group in lysine derivatives of dipeptides and tripeptides [40]. As it was proposed by Harrison et al. [23] the free N-terminal amine group is required for the formation of the macrocyclic b ion structure in the gas-phase. It was shown that cyclization process is completely blocked by N-terminal acetylation [22] [24] [26] [36]. "
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    ABSTRACT: A study was carried out to examine if the amine (-NH2) group located on the side chains of lysine (K), glutamine (Q), or asparagine (N) residue has any effect on the macrocyclization of b ions even though the N-terminals of the peptides were acetylated. The work utilized the model peptides Ac-KYAGFLVG, Ac-QYAGFLV-NH2, and Ac-NYAGFLV-NH2. The CID mass spectra of b(7) ions originated from these three peptides exhibited that the macrocyclization still occurred for the lysine containing peptide in spite of the N-terminal of the peptide was acetylated, but was failed to be observed for glutamine and asparagine containing peptides. These current results reveal that the lysine side chain epsilon-amine group has been involved in the macrocyclization of the peptide b ions for the N-terminal acetylated peptides and consequently, non-direct sequence b ions were observed in the CID mass spectra. However, due to the amide group on the side chains of the glutamine and asparagine residues, the nucleophilicity of their groups greatly reduced; therefore the scrambling b ions were not detected in their b(7) ion CID mass spectra. In addition, the effect of the lysine position was also studied for series of six isomeric octapeptides such as, Ac-KYAGFLVG, Ac-YKAGFLVG, Ac-YAKGFLVG, Ac-YAGKFLVG, Ac-YAGFKLVG and Ac-YAGFLKVG in order to examine the relationship between the intensities of non-direct sequence b ions and the lysine position in the octapeptide series. The results clearly demonstrated that the most abundant non-direct sequence b ions were observed for the first position of lysine residue in the N-terminal acetylated octapeptide, however, when the lysine residue gets closer to the C-terminal position the relative intensities of the scrambled b ions were greatly decreased. (c) 2011 Elsevier B.V. All rights reserved.
    International Journal of Mass Spectrometry 05/2012; 316-318:84-90. DOI:10.1016/j.ijms.2011.12.008 · 1.97 Impact Factor
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